The health of the oral cavity is maintained by salivary secretions. The principal function of salivary glands is to produce these complex fluids. We utilize in vitro dispersed cells of salivary glands, in vivo cannulated glands, and cultured salivary cell lines as laboratory models to understand mechanisms controlling saliva formation. We have focused most of our studies on neurotransmitter regulation of secretory events. During this reporting period the primary focus of signaling studies continues to be muscarinic receptors (mAChRs) in rat parotid gland acinar cells. In parotid cells, stimulation of mAChRs results in the generation of inositol phosphates via the activation of phospholipase C. Subsequently this response leads to the elevation of cytosolic Ca2+ levels and fluid secretion. We have continued to characterize mAChRs in intact rat parotid cells using the binding of a subtype non-selective antagonist (NMS, N-methylscopolamine). We have determined that a moderate population (approximately 30-40%) of spare receptors exist for inositol trisphosphate formation. We have continued in vivo studies of mAChRs in exocrine glands using iodinated QNB (quinuclidinyl benzilate) enantiomers and phamacokinetic analyses. These experiments have led to the development of a clinical research protocol to examine mAChRs in normal human volunteers. To understand how salivary glands transport water we have begun studies on a putative water channel, CHIP28. We have isolated a cDNA encoding a CHIP28-like protein from a rat parotid library and examined its cellular distribution in this gland by in situ hybridization. We have also initiated efforts to transfer foreign genes into rat salivary glands in vivo using replication deficient recombinant adenovirus (Ad) vectors (e.g. containing genes encoding E. Coli beta-galactosidase, beta gal; and human alpha 1 antitrypsin, alpha1AT). Two days after retrograde duct instillation of Ad-beta gal striking expression is seen in acinar and ductal cells of all major salivary glands. Transfer of the alpha1AT gene results in secretion of this protein in gland saliva for 4-10 days.